The characterization of biological compounds is an inherent necessity of any endeavor that seeks to understand life, the processes that sustain life, and the events and elements that affect those processes. Typically, the understanding of life""s processes, and efforts at their control, focuses first at the basic building blocks of life, namely the macromolecular compounds and complexes that differentiate living organisms from mere lifeless primordial ooze. Of particular interest in the understanding and control of life processes are the nucleic acids and the proteins they encode.
In the case of proteins, many characterization methods have remained largely unchanged for decades. For example, current protein characterization methods typically rely, at least in part, upon sodium dodecylsulfate polyacrylamide gel electrophoresis, or SDS-PAGE, to characterize proteins by their relative molecular weights. These methods employ a slab or sheet of cross-linked polyacrylamide. Proteins to be separated and characterized are mixed with a detergent buffer (SDS) and are placed at one edge of the slab, typically in a well. An electric field is applied across the slab, drawing the highly charged detergent micelle containing the proteins through the gel. Larger proteins move through the slab gel more slowly than the smaller proteins, thereby separating out from the greater micelle. After the separation, the gel is contacted with a stain, typically xe2x80x9ccoomassie bluexe2x80x9d or a silver complexing agent, which binds to the different proteins in the gel. In the case of coomassie blue stained gels, the slab gel must be destained to remove the excess stain. These processes result in a ladder of different proteins in the slab gel, separated by size. Silver staining methods are similarly time consuming, and generally yield qualitatively, although non-quantitatively stained gels. Improvements to these processes have produced smaller gels that are faster to run, gels that are purchased xe2x80x9cready-to-use,xe2x80x9d and alternate staining processes. However, he basic SDS-PAGE process has remained largely unchanged as a method of protein characterization.
A number of attempts have been made to apply advances made in other areas to protein characterization. For example, capillary electrophoresis methods, which have proven successful in the analysis of nucleic acids have been attempted in the characterization of proteins. While these methods have proven capable at separating proteins, differences in available labeling chemistries, as well as fundamental structural and chemical differences between proteins and nucleic acids have created substantial barriers to the wide spread use of CE methods in protein characterization. In particular, detection of separated proteins traveling through a capillary has typically required the covalent attachment of a labeling group to all of the proteins, using relatively complex chemistry. Further, the presence of SDS in protein separations, which ensures size based separations, creates further difficulties in both labeling and separation within capillary systems.
It would be desirable to provide methods, devices, systems and kits for characterizing proteins and polypeptides, which would have enhanced throughput, sensitivity and lower space, time and reagent requirements. The present invention meets these and a variety of other needs.
In one aspect, the present invention provides methods of performing an analytical operation on a fluid first sample material. The methods typically comprise providing a microfluidic device that has a body having at least a first channel disposed therein. The first channel comprises first and second channel segments, where the first channel segment comprises a first fluid environment compatible with the performance of a first operation. The first sample material is flowed through the first channel segment to perform the first operation. It is then flowed from the first channel segment into the second channel segment. A first diluent is flowed into the second channel segment, whereby the diluent produces a second fluid environment within the second channel segment, the second environment being more compatible than the first environment with the second operation.
In a related aspect, the invention provides devices for performing analytical operations on sample materials. The devices generally comprise a body structure having a first channel segment disposed within an interior portion of the body, the first channel segment containing a first environment. The device also includes a second channel segment disposed in the body and fluidly connected to the first channel segment. At least a first diluent source is also provided fluidly coupled to the second channel segment. The devices also typically include a flow controller operably coupled to the first diluent source for delivering the first diluent into the second channel segment to provide a second environment within the second channel segment.
In another aspect, the present invention provides a method of characterizing a polypeptide, comprising providing a first capillary channel having a separation buffer disposed within. The separation buffer comprises a polymer matrix, a buffering agent, a detergent, and a lipophilic dye. The polypeptide is introduced into one end of the capillary channel. An electric field is applied across a length of the capillary channel which transports polypeptides of different sizes through the polymer matrix at different rates. The polypeptide is then detected as it passes a point along the length of the capillary channel.
Another aspect of the present invention is a device for separating polypeptides. The device is comprised of a body structure having at least a first capillary channel containing separation buffer within. The separation buffer is comprised of a polymer matrix, a buffering agent, a detergent, and a lipophilic dye capable of binding to the polypeptide or polypeptides. A port disposed in the body structure is in fluid communication with the first capillary channel in order to introduce polypeptides into the first capillary channel.
A further aspect of the present invention is a kit for use in characterizing a polypeptide. The kit is comprised of a microfluidic device hat comprises the elements of the devices described above. The separation buffer is comprised of a polymer matrix, a buffering agent, and a lipophilic dye. Each packaging contains the body structure, the separation buffer, and the lipophilic dye.
Another aspect of the present invention is a system for characterizing a polypeptide. The system includes a body structure having at least a first capillary channel containing a separation buffer disposed therein. The separation buffer is comprised of a polymer matrix, a buffering agent, a detergent, and a lipophilic dye. An electrical power source is operably coupled to opposite ends of the first capillary channel in order to apply an electric field across a length of the capillary channel. A detector is disposed in sensory communication with the capillary channel at a first point to detect the polypeptide as it passes the first point.